The studies planned for the next period of support fall into four main areas: 1) detailed analysis of specific factors and pathways; 2) examination of eukaryotic cell responses to V.cholerae, and V.cholerae responses to the host; 3) development of chip based methods for understanding the role of vibriophages in horizontal gene transfer, and 4) establishment of a defined collection of transposon insertion mutants in most nonessential V.cholerae genes, and their use in functional genomics. Examples of projects include the following: Quorum sensing- our results show that quorum sensing affects the expression of known virulence genes, and biofilm formation. We will use genetic analysis to determine the relative contribution of TCP and biofilm-related genes to colonization, and to explore the possibility of exploiting the quorum sensing pathway to develop novel therapies against cholera. Emergence- we will use genetic and biochemical approaches to assess the contribution to virulence of genes present in two islands found preferentially in 7th pandemic El Tor and O139 strains. These include the "patatin-like" gene, which encodes a homolog of a recently identified phospholipase and may therefore act as a toxin, and the "deoxycytodylate deaminase-like"gene that may increase the fitness of 7th pandemic and O139 strains by enhancing their adaptability through control of mutagenic rates. Expression profiling- we will take advantage of the availability of increasingly accurate human genome microarrays to analyze the changes that occur in human cultured cells after exposure toV. cholerae or its purified products. Evolution- we have shown that horizontal transfer of genetic material via vibriophages likelyplays a major role in the evolution of pathogenic V.cholerae strains. We intend to develop a DNA chip-based method for identifying genes that are selectively packaged into phage particles. Functional genomics- substantially decreased DNA sequencing costs have made possible the construction of a defined collection of transposon insertion mutants in all nonessential V.cholerae genes. Whilethese data will be useful for antibiotic target discovery, we also anticipate that the availability of defined mutants will rapidly advance functional genomic studies in V.cholerae through genetic phenotyping and expression analysis.